Bioelectric hybrid system for producing lactic acid from carbon dioxide

Abstract

A bio-electro-hydraulic system for producing lactic acid from carbon dioxide is disclosed. Recombinant microorganisms can use carbon dioxide and formic acid produced electrochemically from carbon dioxide as carbon sources to produce lactic acid, and thus can be used to convert carbon dioxide into a variety of high-value-added compounds.

Description

Technical Field

[0001] The present invention relates to a bioelectric hybrid system for producing lactic acid from carbon dioxide, and more specifically, to a bioelectric hybrid system for producing lactic acid from carbon dioxide, the system being based solely on a combination of two: an electrode system for electrochemically producing formic acid from carbon dioxide, and a lactic acid fermentation system using recombinant microorganisms that synthesize lactic acid from carbon dioxide and formic acid. Background Technology

[0002] Related technical descriptions The use of fossil fuels has led to increased carbon dioxide emissions, becoming a major cause of climate change. To address this issue, technologies for converting carbon dioxide into beneficial compounds are crucial for building sustainable industries. In particular, electrochemical carbon dioxide reduction (ECO2R) has attracted considerable attention due to its high conversion efficiency and scalability, but it is limited to primarily producing simple C1 and C2 compounds. Improving selectivity to produce compounds with more complex structures remains a technological challenge. Furthermore, biochemical carbon dioxide conversion can produce a variety of compounds under mild conditions, but its efficiency is limited due to issues such as low carbon dioxide solubility and additional energy consumption.

[0003] Bioelectrochemical hybrid systems are considered an innovative alternative for carbon dioxide conversion. These systems provide a flexible platform for producing a variety of compounds based on a combination of electrochemical methods and microorganisms. In particular, formic acid (FA) produced electrochemically has attracted attention due to its high production efficiency and high water solubility. Formic acid can be fed into microbial systems and converted into compounds with more complex structures. In previous studies, the inventors have demonstrated the use of hookworm copper-boring bacteria (… Cupriavidus necator The bioelectric hybrid system can directly produce polyhydroxybutyrate from carbon dioxide (Korean Patent No. 2,000,755). Furthermore, the inventors have constructed a recombinant microorganism capable of growing only in the presence of carbon dioxide and formic acid by introducing and enhancing a metabolic pathway for the synthesis of pyruvate from carbon dioxide and formic acid (Korean Patent No. 2,2690858).

[0004] Meanwhile, lactic acid is a key precursor to biodegradable polylactic acid (PLA) and an important industrial compound used in environmentally friendly plastics and various organic synthesis applications. Producing lactic acid solely from carbon dioxide opens up new possibilities for converting carbon dioxide into high-value chemicals, representing a significant breakthrough in sustainable and environmentally friendly carbon dioxide conversion and the production of carbon-based compounds.

[0005] Therefore, as a result of a significant effort to develop the first bio-electric hybrid system capable of directly producing lactic acid through carbon dioxide conversion, the inventors discovered that when a CO2 electrolyzer for producing formic acid from carbon dioxide is combined with recombinant microorganisms metabolically modified to convert formic acid and carbon dioxide into lactic acid, lactic acid can be produced efficiently using only carbon dioxide as a raw material. Based on this discovery, the present invention was completed. Summary of the Invention

[0006] Therefore, the present invention is made in response to the above-mentioned problems, and one object of the present invention is to provide a recombinant microorganism capable of producing lactic acid from carbon dioxide and formic acid.

[0007] Another object of the present invention is to provide a method for producing lactic acid from formic acid and carbon dioxide using the recombinant microorganism.

[0008] Another object of the present invention is to provide a bioelectric hybrid system device that, by combining recombinant microorganisms with an electrode system for the electrochemical production of formic acid from carbon dioxide, is capable of producing lactic acid solely from carbon dioxide.

[0009] According to one aspect of the invention, the above and other objectives can be achieved by providing a recombinant microorganism capable of producing lactic acid from carbon dioxide and formic acid, wherein a gene encoding L-lactate dehydrogenase is introduced or amplified in a host microorganism having a central carbon assimilation pathway.

[0010] According to another aspect of the present invention, a method for producing a beneficial substance is provided, comprising: (a) culturing recombinant microorganisms using formic acid and carbon dioxide as carbon sources to produce the beneficial substance; and (b) recovering the produced beneficial substance.

[0011] According to another aspect of the present invention, a method for producing lactic acid from carbon dioxide using a bioelectric hybrid system is provided, the method comprising: (a) converting carbon dioxide into formic acid by an electrochemical reduction reaction; and (b) fermenting microorganisms capable of biosynthesizing lactic acid from formic acid and carbon dioxide using the formic acid converted in step (a) to produce lactic acid.

[0012] According to another aspect of the invention, a bio-electro-hydraulic system apparatus for producing lactic acid from carbon dioxide is provided, comprising: (i) an electrolytic reactor for converting carbon dioxide into formic acid via an electrochemical reduction reaction; and (ii) a biosynthetic reactor fluidly connected to the electrolytic reactor for producing lactic acid from formic acid by fermentation of microorganisms in an electrode solution containing formic acid.

[0013] Beneficial effects According to the present invention, the recombinant microorganisms can use carbon dioxide and formic acid produced by the electrochemistry of carbon dioxide as carbon sources to produce lactic acid, and thus can be used to convert carbon dioxide into a variety of high-value-added compounds. Attached Figure Description

[0014] The above and other objects, features and advantages of the present invention will become more apparent from the following detailed description taken in conjunction with the accompanying drawings, in which: Figure 1 (a) is a schematic diagram illustrating the core metabolic pathway by which recombinant microorganisms produce lactic acid using carbon dioxide and formic acid (FA), as well as the genes, enzymes, and metabolites involved in this pathway. Figure 1 (b) shows cell growth and lactic acid (LA) production measured when recombinant microorganisms were cultured in flasks using carbon dioxide, formic acid (FA), and glucose (Glc) as carbon sources; and Figure 1 (c) shows cell growth and lactic acid (LA) production measured when the recombinant microorganism was cultured in a fermenter using only formic acid and carbon dioxide as carbon sources; Figure 2 This is a schematic diagram illustrating the plasmid p518FA1_Strepto_ldhA, which was constructed by inserting the following genes involved in carbon dioxide and formic acid assimilation: derived from the genus Candida ( Candida Formate dehydrogenase, derived from Arabidopsis thaliana ( Arabidopsis Formate dehydrogenase mutants and those derived from Streptococcus spp. Streptococcus The lactate dehydrogenase gene; Figure 3 The cell growth of recombinant microorganism FC15 (a) and parental strain AK01 (b) at high cell density in the presence of carbon dioxide and formic acid only is shown. 13 C-labeled glucose was pre-cultured, and in a fermenter with only formic acid and carbon dioxide as carbon sources, the pre-culture and fermenter cultures showed differences. 13 C-labeled lactate ratio (c); Figure 4 (a) is a schematic diagram illustrating a bioelectric hybrid system according to the present invention. Figure 4 (b) shows the production of formic acid under different current densities in a physiologically compatible negative electrode solution; Figure 5 The diagram illustrates lactic acid production using only carbon dioxide in a bioelectric hybrid system according to the invention, with different initial seeding cell densities, wherein (a) and (b) have an initial seeding cell density of 1.5, (c) and (d) have an initial seeding cell density of 3.3, and (e) and (f) have an initial seeding cell density of 5.0; and Figure 6 The following diagram illustrates lactic acid production using only carbon dioxide in a bioelectric hybrid system under different current density conditions, where (a) and (b) have a current density of -90 mA cm⁻¹. -2 (c) and (d) have a current density of -105 mA cm⁻¹ -2 (e) and (f) have a current density of -120 mA cm⁻¹ -2 Furthermore, (g) and (h) have a current density of -140 mA cm⁻¹. -2 . Detailed Implementation

[0015] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as understood by one of ordinary skill in the art to which this invention pertains. Generally, the nomenclature used herein is well-known and commonly used in the field.

[0016] Formic acid (FA) is a C1 compound that can be efficiently converted into carbon dioxide via electrochemical conversion. Formic acid is liquid at room temperature, making it easy to store and transport. Furthermore, formic acid can be used as a reducing agent for cellular carbon source assimilation via formate dehydrogenase (Fdh). Due to these advantages, formic acid has attracted attention as a carbon source for microbial metabolism. In previous studies, the inventors designed and validated a novel metabolic pathway for converting formic acid (a C1 compound) into beneficial multi-carbon compounds (Korean Patent No. 2,000,755). Furthermore, by introducing and enhancing a metabolic pathway for the synthesis of pyruvate from carbon dioxide and formic acid, the inventors constructed a recombinant microorganism capable of growth solely on carbon dioxide and formic acid (Korean Patent No. 2,690,858).

[0017] In this invention, recombinant microorganisms (Korean Patent No. 2,690,858) capable of growing only in the presence of carbon dioxide and formic acid were further metabolically engineered. The inventors also improved a culture medium and cultivation method to integrate them with an electrode system for formic acid production, and discovered that the improved recombinant *E. coli* ( Escherichia coli It can produce lactic acid from carbon dioxide alone.

[0018] In the recombinant microorganism constructed in Korean Patent No. 2,690,858, formic acid and carbon dioxide were introduced into the central metabolic pathway in the form of pyruvate. To produce lactate from formic acid and carbon dioxide based on this recombinant microorganism (parental strain), a lactate dehydrogenase that converts pyruvate to lactate was introduced. Furthermore, endogenous lactate dehydrogenase genes (e.g., from *Escherichia coli*) were also incorporated. E. coli In ) ldhA , dld and lldDLactic acid production from pyruvate may be induced under low pH and anaerobic conditions, and may also convert the produced lactic acid to pyruvate in the reverse reaction. For this reason, these genes were removed. Additionally, the production of lactic acid from pyruvate was improved by introducing a gene encoding L-lactate dehydrogenase from Streptococcus.

[0019] Therefore, in one aspect, the present invention relates to a recombinant microorganism capable of producing lactic acid from carbon dioxide and formic acid, wherein a gene encoding L-lactate dehydrogenase is introduced or amplified in a host microorganism having a central carbon assimilation pathway.

[0020] The recombinant microorganism is characterized by the deletion of the gene encoding endogenous lactate dehydrogenase.

[0021] In this invention, L-lactic acid dehydrogenase can be an enzyme capable of converting pyruvate into lactate.

[0022] As used in this article, the term "central carbon assimilation pathway" refers to a metabolic pathway involved in the assimilation of central carbons (such as formic acid and / or carbon dioxide).

[0023] Figure 1 This is a schematic diagram illustrating key metabolic pathways involved in the assimilation of carbon dioxide and formic acid in recombinant microorganisms, as well as the genes, enzymes, and metabolites involved. In this invention, the formic acid assimilation pathway is a cycle in which microorganisms assimilate carbon dioxide and formic acid into pyruvate, which contains three carbon atoms. Figure 1 The genes, coenzymes, and energy transfer substances involved in the formic acid assimilation pathway in Escherichia coli are illustrated.

[0024] In one embodiment of the present invention, a recombinant microorganism was constructed by deleting the endogenous lactate dehydrogenase gene from a host microorganism with formic acid and carbon dioxide assimilation pathways and introducing a gene encoding a heterologous L-lactate dehydrogenase capable of converting pyruvate in the host microorganism into lactate.

[0025] In this invention, the formic acid assimilation pathway can combine with the central carbon assimilation pathway to synthesize carbon compounds with C3 or higher carbon atoms. The host microorganism can possess: i) an endogenous central carbon assimilation pathway or ii) an exogenous central carbon assimilation pathway.

[0026] The gene encoding l-lactate dehydrogenase involved in lactate production may be a gene encoding l-lactate dehydrogenase from the genus Streptococcus, which may be involved in the production of lactate from pyruvate, and may include the base sequence represented by SEQ ID NO: 6.

[0027] In this invention, the recombinant microorganism may be characterized by the deletion of the host cell's genes encoding lactate dehydrogenase (ldhA, dld, lldD).

[0028] The host cell's genes encoding lactate dehydrogenase (ldhA, dld, lldD) can be ldhA, dld, or lldD genes. The lldD gene is removed to prevent the redistribution and reuptake of l-LA, while the dld gene, which expresses d-LA production capacity, is removed to improve l-LA selectivity.

[0029] The recombinant microorganisms of the present invention may further include genes encoding at least one selected from the group consisting of: formate-tetrahydrofolate ligase, methylenetetrahydrofolate cyclic hydrolase, methylenetetrahydrofolate dehydrogenase, formate dehydrogenase, and formate dehydrogenase mutants.

[0030] The gene encoding formate-tetrahydrofolate ligase may include the sequence of SEQ ID NO: 1, the gene encoding methylenetetrahydrofolate cyclic hydrolase may include the sequence of SEQ ID NO: 2, the gene encoding methylenetetrahydrofolate dehydrogenase may include the sequence of SEQ ID NO: 3, and the gene encoding formate dehydrogenase may include the sequence of SEQ ID NO: 4. Alternatively, the gene encoding a formate dehydrogenase mutant may include the sequence of SEQ ID NO: 5 (Table 1).

[0031] [Table 1] A gene encoding at least one of the group consisting of formate-tetrahydrofolate ligase, methylenetetrahydrofolate cyclic hydrolase, methylenetetrahydrofolate dehydrogenase, formate dehydrogenase, and formate dehydrogenase mutants may be introduced by cloning into a vector having a copy number of 1 to 12 origins of replication, preferably by cloning into a vector having a copy number of 1 to 5 origins of replication, but not limited thereto.

[0032] The host microorganism can be free Escherichia coli (Escherichia coli spp.) Escherichia ), Mannella spp. Mannheimia ), Rhodomycium genus ( Rhodobacter ) and Methylobacterium ( Methylobacterium Groups consisting of, but not limited to, ().

[0033] The recombinant microorganisms according to the present invention use only formic acid and carbon dioxide as carbon sources to produce lactic acid.

[0034] The genes of the present invention can be modified in various ways in the coding region, as long as the amino acid sequence of the protein expressed by the coding region does not change; they can also be freely mutated or modified, as long as they do not affect the expression of genes in regions outside the coding region, and such mutated or modified genes are also within the scope of the present invention.

[0035] Therefore, the present invention also includes polynucleotides having substantially the same nucleotide sequence as genes and gene fragments. The term "substantially the same polynucleotide" means a gene encoding an enzyme having the same function as the enzyme used in the present invention, regardless of sequence homology. The term "gene fragment" also means a gene encoding an enzyme having the same function as the enzyme used in the present invention, regardless of the length of the fragment.

[0036] Furthermore, as long as the titer and activity of the corresponding enzyme are not affected, the amino acid sequence of the protein that is the expression product of the gene of the present invention can be obtained from biological resources (such as various microorganisms), and these biological resources are also within the scope of the present invention.

[0037] Therefore, the present invention also includes polypeptides having substantially the same amino acid sequence as the protein, and fragments of polypeptides. The term "substantially the same polypeptide" means a protein having the same function as the protein used in the present invention, regardless of amino acid sequence homology. The term "polypeptide fragment" also means a protein having the same function as the protein used in the present invention, regardless of fragment length.

[0038] As used herein, the term "vector" refers to a DNA product containing a DNA sequence operatively linked to a regulatory sequence capable of expressing DNA in a suitable host, and can be a plasmid, phage particle, or a simple potential genomic insert. Once transformed into a suitable host cell, the vector can replicate and function independently of the host genome, or in some cases, integrate into the host genome. Since plasmids are the most commonly used type of vector, the terms "plasmid" and "vector" are sometimes used interchangeably in this specification. For the purposes of this invention, plasmid vectors are preferred. Typical plasmid vectors suitable for this purpose include: (a) an origin of replication for efficient replication, thereby including several to hundreds of plasmid vectors per host cell; (b) an antibiotic resistance gene for screening host cells transformed with the plasmid vector; and (c) a restriction endonuclease cleavage site for inserting a foreign DNA fragment therein. Even if a suitable restriction endonuclease cleavage site is not available, the vector and foreign DNA can be readily ligated using synthetic oligonucleotide adaptors or adapters according to conventional methods. After ligation, the vector should be transformed into a suitable host cell. The conversion can be easily carried out using the calcium chloride method or electroporation (Neumann, et al., EMBO J., 1: 841, 1982).

[0039] In this invention, "gene reduction (or inactivation)" means that the expression of the gene is reduced compared to the wild type, or that the function or activity of the protein encoded by the gene is reduced compared to the wild type.

[0040] Expression vectors known in the art can be used as vectors for enhancing or overexpressing genes according to the present invention.

[0041] When a nucleotide sequence is aligned with another nucleotide sequence based on its functional relationship, it is said to be "operably linked". One or more genes and one or more regulatory sequences can be linked in this way so that gene expression can occur when a suitable molecule (e.g., a transcription activator protein) is linked with one or more regulatory sequences. For example, when expressing a proprotein involved in polypeptide secretion, the DNA of the prosequence or secretion leader sequence is operably linked with the DNA of the polypeptide; when a promoter or enhancer affects the transcription of a sequence, the promoter or enhancer is operably linked with the coding sequence; when a ribosome binding site affects the transcription of a coding sequence, the ribosome binding site is operably linked with the coding sequence; or when the ribosome binding site is positioned to facilitate translation, the ribosome binding site is operably linked with the coding sequence. Generally, "operably linked" means that the linked DNA sequence is in contact with it, or that the secretion leader sequence is in contact with it and is present within the reading frame. However, enhancers do not need to be in contact. The ligation of these sequences is performed by linking (ligation) at convenient restriction endonuclease sites. When such sites are not available, synthetic oligonucleotide adaptors or linkers according to conventional methods are used.

[0042] As is known in the art, in order to enhance the expression level of a transgene in a host cell, the gene should be operatively linked to a transcriptional / translational expression regulatory sequence that functions in a selected expression host. Preferably, the expression regulatory sequence and the corresponding gene are contained in a recombinant vector containing both a bacterial selection marker and an origin of replication. When the host cell is a eukaryotic cell, the recombinant vector should further include an expression marker useful in a eukaryotic expression host.

[0043] Host cells transformed with the aforementioned recombinant vector constitute another aspect of the present invention. As used herein, the term "transformation" means the introduction of DNA into a host and the use of extrachromosomal factors or chromosomal integration to make the DNA replicable.

[0044] It should be understood that not all vectors function identically when expressing the DNA sequences of this invention. Similarly, not all hosts function identically for the same expression system. However, those skilled in the art can make appropriate selections from a variety of vectors, expression regulatory sequences, and hosts without conducting excessive experimentation or departing from the scope of this invention. For example, the selection of a vector should take into account the host, as the vector should replicate within the host. The number of replications of the vector, its ability to control the number of replications, and the expression of other proteins encoded by the corresponding vector (such as the expression of antibiotic markers) should also be considered.

[0045] Furthermore, the genes introduced in this invention can be introduced into the genome of a host cell and can exist as chromosomal factors. It will be apparent to those skilled in the art that inserting genes into the genome of a host cell has the same effect as introducing a recombinant vector into the host cell.

[0046] The aforementioned recombinant microorganisms can produce lactic acid using pyruvate assimilated from formic acid and carbon dioxide.

[0047] The host microorganism can be selected from, but is not limited to, the group consisting of, for example, Escherichia coli, Mansonia spp., Rhodotorula spp., and Methylobacterium spp.

[0048] The resulting recombinant plasmid was used to transform *E. coli* to produce recombinant *E. coli*. The *E. coli* strain used in this invention is *E. coli* DH5α (Invitrogen, USA), and the transformation was performed using chemical transformation methods commonly used in the art.

[0049] Meanwhile, this invention shows that, under conditions of carbon dioxide, formic acid, and glucose, and in a culture medium that provides only carbon dioxide and formic acid as carbon sources, the recombinant microorganism can produce 4.39 g / L and 25.5 mg / L of lactic acid, respectively.

[0050] Therefore, in another aspect, the present invention relates to a method for producing a beneficial substance, the method comprising: (a) Using formic acid and carbon dioxide as carbon sources to culture recombinant microorganisms to produce beneficial substances; and (b) Recycle the beneficial substances produced.

[0051] In this invention, the beneficial substance may be lactic acid.

[0052] In another aspect, the present invention relates to a method for producing lactic acid from carbon dioxide using a bioelectric hybrid system, the method comprising: (a) Converting carbon dioxide into formic acid via an electrochemical reduction reaction; and (b) Using the formic acid converted in step (a), fermenting microorganisms capable of biosynthesizing lactic acid from formic acid and carbon dioxide to produce lactic acid.

[0053] In this invention, in step (a), carbon dioxide is converted into formic acid at the negative electrode via an electrochemical reduction reaction, and in step (b), fermentation is carried out in the presence of an electrode solution containing the formic acid converted in step (a), which may be a negative electrode solution.

[0054] In this invention, the concentration of formic acid in the negative electrode solution can be from 0.1 to 10 g / L.

[0055] In this invention, the electrode solution may contain a phosphoric acid compound, and the concentration of the phosphoric acid compound in the electrode solution may be 10 to 40 g / L.

[0056] In this invention, the phosphoric acid compound may include at least one selected from the group consisting of: potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), and tripotassium phosphate (K3PO4).

[0057] In this invention, the microorganisms capable of biosynthesizing lactic acid from carbon dioxide and formic acid can be recombinant microorganisms.

[0058] In another aspect, the present invention relates to a bioelectric hybridization system apparatus for producing lactic acid from carbon dioxide, comprising: (i) an electrolytic reactor for converting carbon dioxide into formic acid via an electrochemical reduction reaction; and (ii) A biosynthetic reactor fluidly connected to the electrolytic reactor, wherein lactic acid is produced from formic acid by microbial fermentation in an electrode solution containing formic acid.

[0059] Figure 4 (A) is a schematic diagram illustrating a bioelectric hybrid system device utilizing electrochemical carbon dioxide reduction and microbial fermentation according to an embodiment of the present invention.

[0060] The bio-electro-hybrid system may further include a first conduit that fluidly connects the electrolysis reactor to the biosynthesis reactor and delivers a negative electrode solution containing formic acid from the electrolysis reactor to the biosynthesis reactor.

[0061] Furthermore, the bio-electric hybrid system may further include a second conduit that fluidly connects the biosynthetic reactor to the electrolytic reactor and delivers the electrode solution within the biosynthetic reactor to the electrolytic reactor.

[0062] An electrolytic reactor may include a gas chamber, a negative electrode solution chamber, a positive electrode solution chamber, a gas diffusion electrode, a cation exchange membrane, and a catalyst. Specifically, the electrolytic reactor may include a gas diffusion electrode disposed between the gas chamber and the cathode solution chamber, and a cation exchange membrane disposed between the negative electrode solution chamber and the positive electrode solution chamber. Furthermore, the negative electrode solution chamber may include a reference electrode, and a counter electrode may be installed outside the positive electrode solution chamber.

[0063] The gas chamber can serve as a channel for carbon dioxide gas to flow to the gas diffusion electrode, the negative electrode solution chamber can serve as a channel for the negative electrode solution to flow through, and the positive electrode solution chamber can serve as a channel for the positive electrode solution to flow through.

[0064] A gas diffusion electrode is a porous electrode that ensures gas permeability to promote gas-related electrode reactions by allowing contact between the electrode (solid phase), electrode solution (liquid phase), and gas phases. The gas diffusion electrode can be hydrophobic to prevent electrode solution leakage and can simultaneously serve as the negative electrode and working electrode in an electrolytic reactor. Furthermore, the gas diffusion electrode can provide gaseous carbon dioxide near the active sites of the electrocatalyst, thereby promoting high current density operation of the electrochemical carbon dioxide reaction.

[0065] At the positive electrode, a water splitting reaction occurs to produce hydrogen ions and oxygen. The hydrogen ions generated at the positive electrode can migrate through the cation exchange membrane to the gas diffusion electrode and are then supplied to the gas diffusion electrode for the production of formic acid.

[0066] At the gas diffusion electrode, which serves as the negative electrode, carbon dioxide supplied from the gas chamber can be converted into formic acid through an electrochemical reduction reaction with hydrogen ions generated from water decomposition at the positive electrode. The negative electrode solution containing formic acid converted in this manner can be introduced from the electrolysis reactor into the biosynthesis reactor through a first pipe, and can serve as nutrients for the microorganisms present in the biosynthesis reactor to synthesize lactic acid.

[0067] Under favorable environmental reaction conditions, the electrochemical reduction of formic acid exhibits high selectivity, thereby maximizing the utilization of carbon dioxide and providing an environmentally beneficial method.

[0068] The concentration of formic acid in the negative electrode solution can range from 0.1 to 10 g / L, preferably from 0.3 to 6 g / L, and more preferably from 0.5 to 3 g / L. When the formic acid concentration is below this range, microorganisms may have difficulty obtaining a carbon source and instead digest lactic acid instead of formic acid. When the concentration exceeds this range, the toxicity of formic acid may affect microbial metabolism.

[0069] Cation exchange membranes reduce the permeation of organic matter (such as substrates that serve as nutrients for microorganisms at the positive electrode), thereby preventing organic contamination of the negative electrode. Furthermore, cation exchange membranes reduce the permeability of metal cations and increase the permeability of hydrogen ions, thus preventing a decrease in the pH of the positive electrode and improving the efficiency of the electrolysis system while maintaining stable efficiency.

[0070] Catalysts can be noble metal catalysts, non-noble metal catalysts, or organometallic catalysts. Alternatively, these catalysts can be used in catalyst structures dispersed on various supports. Examples of noble metal catalysts include Pt, Ru, Ir, Rh, Pd, Au, Ag, and Re, while examples of non-noble metal catalysts include Fe, Co, Ni, Mn, Mo, Zn, V, Cr, Cu, Al, Ga, Ge, In, Sn, and Sb. A representative example of a non-noble metal catalyst is Sn. Because Sn is abundant and non-toxic on Earth, Sn catalysts can be used for CO2 reduction reactions.

[0071] Examples of organometallic catalysts include Fe-based, Co-based, Ni-based, Mn-based, Cu-based, and Sn-based organometallic catalysts. These catalysts can be used alone or as alloys. For example, alloy-type organometallic catalysts include noble metal-based binary alloy catalysts, platinum-transition metal-based binary alloy catalysts, platinum-organometallic binary alloy catalysts, or multi-component alloy catalysts.

[0072] These metal catalysts can be used directly or supported on a carrier. These carriers can be carbon-based materials such as graphite, Denka Black, Ketjen Black, acetylene black, carbon nanotubes, carbon nanofibers, carbon nanowires, carbon nanospheres, or activated carbon. Alternatively, the carrier can be inorganic particles such as alumina, silica, zirconium oxide, or titanium dioxide. However, carbon-based materials are commonly used.

[0073] Next, the biosynthetic reactor contains microorganisms that synthesize lactic acid and electrode solutions for microbial growth.

[0074] In embodiments of the present invention, the electrode solution in which microorganisms can survive contains 2.1 g / L KH2PO4, 39 g / L K2HPO4, 0.5 g / L MgSO4, 0.8 g / L citric acid, 0.012 g / L IPTG and 2 g / L NH4Cl, but is not limited thereto.

[0075] A hollow fiber filter can be installed between the electrolysis reactor and the biosynthesis reactor to separate the microorganisms from the electrode solution before introducing the electrode solution from the biosynthesis reactor into the electrolysis reactor.

[0076] Furthermore, step (b) may include the biosynthesis of lactic acid via microbial fermentation in an electrode solution mixed with the negative electrode solution containing formic acid from step (a). The initial inoculum size of the microorganisms may be OD. 600 2 to OD 600 Cell density of 10, but not limited to this.

[0077] Furthermore, in step (b), the current density for producing formic acid by electrochemical carbon dioxide reduction in the electrochemical electrolyzer can be -90 mA cm⁻¹. -2 to -140 mA cm -2 However, it is not limited to this.

[0078] The recombinant microorganisms according to the present invention can produce lactic acid using only carbon dioxide and formic acid. Recombinant microorganisms developed by the inventors in previous studies were able to grow on only carbon dioxide and formic acid without a glucose supply, but were still able to produce lactic acid. However, by removing the endogenous lactate dehydrogenase gene and introducing a gene encoding a heterologous lactate dehydrogenase, the recombinant microorganisms according to the present invention are able to produce 25 mg / L of lactic acid using only carbon dioxide and formic acid.

[0079] Furthermore, in this invention, a bioelectric hybrid system, based on a method for producing lactic acid solely from formic acid and carbon dioxide combined with an electrode system for producing formic acid from carbon dioxide, is capable of producing 88.3 mg / L of lactic acid solely from carbon dioxide. This is achieved by optimizing the developed electrode solution, initial microbial inoculum size, and current density.

[0080] Example The present invention will now be described in more detail with reference to the following embodiments. However, it will be apparent to those skilled in the art that the following embodiments are provided for illustrative purposes only and should not be construed as limiting the scope of the invention.

[0081] Example 1: Developing recombinant microorganisms that produce lactic acid from carbon dioxide and formic acid through the modification of metabolically engineered strains. To develop a strain with enhanced assimilation efficiency from carbon dioxide and formic acid alone, the gene encoding lactate dehydrogenase from *E. coli* was developed. ldhA , dld and lldD The enzyme was missing. A heterologous lactate dehydrogenase (ldh) from Streptococcus was introduced. strep The gene was used to develop a recombinant microorganism capable of selectively producing L-lactic acid.

[0082] Specifically, in constructing the gene fragment required for a plasmid to introduce a foreign gene, a heterologous lactate dehydrogenase (ldh) is used. strepUsing microbial genomic DNA of the gene as a template and primers designed for amplification, PCR was performed, and the amplified gene fragment was then recovered and purified. The sequences of the gene and primers are shown in Tables 2 and 3.

[0083] [Table 2] [Table 3] The plasmid backbone gene fragment was prepared by performing PCR using primers, followed by recovery and purification of the amplified gene fragment.

[0084] Using the Gibson assembly method ( Gibson et al., Nat. Methods (References 6:5, 343-345, 2009) This method involves assembling plasmid backbone gene fragments and amplified gene fragments into plasmids, a commonly used approach for assembling gene fragments. Each plasmid is constructed to contain one or more exogenous genes listed in the table provided above. Figure 2 ) [Table 4] Recombinant Escherichia coli were generated by transforming the recombinant plasmid produced by this method. The Escherichia coli strain used in this invention is Escherichia coli DH5α (Invitrogen, USA), and the transformation was performed using chemical transformation methods commonly used in the art.

[0085] In addition, the endogenous lactate dehydrogenase gene present in E. coli... ldhA , dld and lldD Missing.

[0086] [Table 5] The following strains were generated by introducing the p518FA1R_Strepto_ldhA plasmid into the strains produced above.

[0087] [Table 6] To determine the enhanced carbon dioxide and formic acid assimilation capacity of the recombinant Escherichia coli strain, the recombinant Escherichia coli strain was used as the experimental group, and the previously established Escherichia coli strain was used as the control group. Both strains were cultured in flasks in mm9 medium containing formic acid and carbon dioxide. The mm9 medium had the following composition (Table 7).

[0088] [Table 7] To determine the lactic acid production of strain FC15, the strain was first cultured in shake flasks in MM9 medium containing glucose, followed by fermentation in a fermenter using MM9 medium supplemented with formic acid and carbon dioxide as carbon sources. An additional 300 µL of 10% formic acid solution was supplied to the glucose-containing medium to replenish formic acid. Under the culture conditions providing glucose, formic acid, and carbon dioxide, strain FC15 produced 4.39 g / L of lactic acid (…). Figure 1 B). Under fermenter culture conditions providing only carbon dioxide and formic acid as carbon sources, strain FC15 produced 25.5 mg / L of lactic acid (B). Figure 1 C). To evaluate the lactic acid production capacity of the parental strain AKO1, horizontal fermentation was performed using only carbon dioxide and formic acid as carbon sources. At OD... 600 Under initial inoculum conditions of 4.03, 143.5 mg / L of lactic acid was produced, while no lactic acid was detected in strain AKO1. Figure 3 A and Figure 3 B).

[0089] In addition, the pre-fermentation step provides 13 C-labeled glucose as a carbon source, and under conditions where only carbon dioxide and formic acid are provided as carbon sources without... 13 Lactic acid was determined under C-labeled glucose conditions. 13 C-marking ratio. In containing 13 Under C-labeled glucose conditions, approximately 88% of the lactic acid is... 13 C labeling. However, under conditions where only formic acid and carbon dioxide were provided as carbon sources, only about 10% of lactic acid was labeled, similar to the control group. Figure 3 C). These results show that strain FC15 can produce lactic acid from carbon dioxide and formic acid alone.

[0090] Example 2: Development of a bioelectric hybrid system for producing lactic acid from carbon dioxide A bioelectric hybrid system for producing lactic acid from carbon dioxide was developed by combining the lactic acid production method using the recombinant FC15 strain constructed in Example 1 with an electrode system for producing formic acid from carbon dioxide. Figure 4 A).

[0091] To develop this bioelectric hybrid system, a physiologically compatible negative electrode solution capable of generating lactic acid was developed. The physiologically compatible negative electrode solution must possess sufficient ionic conductivity to function as an electrolyte and must be free of trace metal solutions to prevent trace metal deposition on the electrode surface. Furthermore, nitrogen sources must be excluded to prevent solid precipitation.

[0092] However, in the metabolic pathway of strain FC15, glycine serves as a precursor for lactate synthesis. Glycine synthesis requires a nitrogen source, and glycine is subsequently degraded, thus no nitrogen compounds are consumed in the overall metabolic cycle. Figure 1 A). Therefore, a nitrogen source is required via the negative electrode electrolyte. This is achieved by adding 2 g / L of NH4Cl to a conventional dihydrogen phosphate solution. The composition of the developed negative electrode solution is shown in Table 8.

[0093] [Table 8] Using the developed negative electrode solution, at -90 to -140 mA / cm 2 The high current density operation of the CO2 electrolyzer leads to the stable production of formic acid. Figure 4 B).

[0094] This bioelectric hybrid system was operated using a Sn gas diffusion electrode and the FC15 strain. First, a CO2 electrolyzer was run to accumulate formic acid. Then, the FC15 strain was inoculated into the fermenter, and the hybrid system continued to run for 72 hours. t = 0 h indicates the time point at which the FC15 strain was inoculated.

[0095] To determine the optimal cell density for the system, cells at the same growth stage were seeded at different initial cell densities. Experiments were conducted at -120 mA / cm². 2 The following steps will be performed, starting with the initial OD. 600 The doses were 1.5 (stem cell weight 1.74 mg), 3.3 (21.4 mg), and 5.0 (36.6 mg), respectively.

[0096] like Figure 5 As shown, under all conditions, strain FC15 consumed formic acid produced by electrochemical CO2 reduction, with the highest concentration at OD. 600 The highest lactic acid production was observed under a pH of 3.3.

[0097] In the initial OD 600 Under a value of 3.3, different current densities (-90, -105, -120 and -140 mA cm⁻¹) were used. 2 Lactic acid production was observed.

[0098] like Figure 6 As shown, changes in current density lead to changes in the formic acid supply, which in turn affects lactic acid production. At -120 mA cm⁻¹ 2 At the specified current density, a maximum of 88.3 mg / L of lactic acid was produced over 66 hours. Under all conditions, the electrochemical reaction voltage and Faraday efficiency remained stable.

[0099] At -90 mA / cm2 At low current densities, formic acid is completely consumed in the early stages, and the formic acid concentration drops to 0 after 24 hours, resulting in a low lactic acid production of 20 mg / L. At -105 mA / cm² 2 At a current density of -120 mA / cm², a maximum of 68 mg / L of lactate was produced, but it was subsequently degraded as L-LA was used for cellular metabolism. 2 At a current density of -140 mA / cm², lactic acid production is more stable, indicating that the supply of FA is sufficient to prevent lactic acid degradation. However, when the current density increases to -140 mA / cm², lactic acid production becomes more stable. 2 At that time, lactic acid production decreased to 22 mg / L. 1 Furthermore, the concentration of formic acid increases as the pH decreases. Under high concentrations of formic acid, cells cannot effectively convert formic acid into lactic acid due to its toxicity.

[0100] Although specific configurations of the invention have been described in detail, those skilled in the art will understand that these detailed descriptions are provided for illustrative purposes only as preferred embodiments and should not be construed as limiting the scope of the invention. Therefore, the essential scope of the invention is defined by the appended claims and their equivalents.

Claims

1. A recombinant microorganism capable of producing lactic acid from carbon dioxide and formic acid. in, In host microorganisms with a central carbon assimilation pathway, genes encoding L-lactate dehydrogenase are introduced or amplified.

2. The recombinant microorganism according to claim 1, wherein, The host microorganism has either an i) endogenous central carbon assimilation pathway or an ii) exogenous central carbon assimilation pathway.

3. The recombinant microorganism according to claim 1, wherein, The gene encoding L-lactate dehydrogenase is represented by the base sequence of SEQ ID NO:

6.

4. The recombinant microorganism according to claim 1, wherein, The host cell does not possess the endogenous lactate dehydrogenase gene.

5. The recombinant microorganism according to claim 1 further comprises a gene encoding at least one selected from the group consisting of: formate-tetrahydrofolate ligase, methylenetetrahydrofolate cyclic hydrolase, methylenetetrahydrofolate dehydrogenase, formate dehydrogenase, and formate dehydrogenase mutant.

6. The recombinant microorganism according to claim 5, wherein, The gene encoding formate-tetrahydrofolate ligase is represented by the sequence SEQ ID NO:

1. The gene encoding methylenetetrahydrofolate cyclic hydrolase is represented by the sequence SEQ ID NO:

2. The gene encoding methylenetetrahydrofolate dehydrogenase is represented by the sequence SEQ ID NO:

3. The gene encoding formate dehydrogenase is represented by the sequence SEQ ID NO: 4, or The gene encoding the formate dehydrogenase mutant is represented by the sequence SEQ ID NO:

5.

7. The recombinant microorganism according to claim 5, wherein, The gene was introduced into a vector containing a replication origin with a copy number of 1 to 5 by cloning.

8. The recombinant microorganism according to claim 1, wherein, The host microorganism was selected from the group consisting of: Escherichia coli ( Escherichia ), Mannella spp. Mannheimia ), Rhodomycium genus ( Rhodobacter ) and Methylobacterium ( Methylobacterium ).

9. A method for producing a beneficial substance, comprising: (a) Using formic acid and carbon dioxide as carbon sources to culture recombinant microorganisms according to any one of claims 1 to 8 to produce beneficial substances; as well as (b) Recycle the beneficial substances produced.

10. The method according to claim 9, wherein, The beneficial substance is lactic acid.

11. A method for producing lactic acid from carbon dioxide using a bioelectric hybrid system, the method comprising: (a) Converting carbon dioxide into formic acid via an electrochemical reduction reaction; as well as (b) Using the formic acid converted in step (a), fermenting microorganisms capable of biosynthesizing lactic acid from formic acid and carbon dioxide to produce lactic acid.

12. The method according to claim 11, wherein, In step (a), carbon dioxide is converted into formic acid at the negative electrode through an electrochemical reduction reaction.

13. The method according to claim 11, wherein, In step (b), the fermentation is carried out in the presence of an electrode solution containing the formic acid converted in step (a).

14. The method according to claim 13, wherein, The electrode solution in step (b) is a negative electrode solution.

15. The method according to claim 14, wherein, The concentration of formic acid in the negative electrode solution is 0.1 to 10 g / L.

16. The method according to claim 13, wherein, The electrode solution contains a phosphoric acid compound.

17. The method according to claim 16, wherein, The concentration of the phosphoric acid compound in the electrode solution is 10 to 40 g / L.

18. The method according to claim 16, wherein, The phosphoric acid compound includes at least one selected from the group consisting of potassium dihydrogen phosphate (KH2PO4), dipotassium hydrogen phosphate (K2HPO4), and tripotassium phosphate (K3PO4).

19. The method according to claim 11, wherein, The microorganism capable of biosynthesizing lactic acid from carbon dioxide and formic acid is a recombinant microorganism according to any one of claims 1 to 8.

20. A bioelectric hybridization system device for producing lactic acid from carbon dioxide, comprising: (i) An electrolytic reactor for converting carbon dioxide into formic acid via an electrochemical reduction reaction; as well as (ii) A biosynthetic reactor fluidly connected to the electrolytic reactor, wherein lactic acid is produced from formic acid by microbial fermentation in an electrode solution containing formic acid.

21. The bioelectric hybrid system device according to claim 20, wherein, The electrolytic reactor includes a gas diffusion electrode that serves as the negative electrode.

22. The bioelectric hybrid system apparatus according to claim 20, further comprising a first conduit for fluidly connecting the electrolysis reactor to the biosynthesis reactor and for conveying a negative electrode solution containing formic acid from the electrolysis reactor to the biosynthesis reactor.

23. The bioelectric hybrid system apparatus of claim 20, further comprising a second conduit for fluidly connecting the biosynthetic reactor to the electrolytic reactor and for conveying the electrode solution within the biosynthetic reactor to the electrolytic reactor.

24. The bioelectric hybrid system apparatus of claim 20, further comprising a hollow fiber filter for separating the microorganisms from the electrode solution before the culture medium in the biosynthetic reactor is introduced into the electrolytic reactor.

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